Novel process for the fermentative production of cephalosporin

ABSTRACT

A method for the recovery of an N-substituted cephalosporanic acid compound of general formula (I), wherein R 2  is selected from the group consisting of adipyl (1,4-dicarboxybutane), succinyl, glutaryl adipyl, pimelyl, suberyl, 2-carboxyethyithio)acetyl, 3-carboxyethylthio)propionyl, higher alkyl saturated and higher alkyl unsaturated dicarboxylic acids, from a complex mixture comprising in addition to the compound of general formula (I) 6-aminopenicillanic acid (6-APA) and optionally one or more N-substituted penicillanic acid compounds, comprising the steps of: (a) acidifying the complex mixture to a pH below 6.5 and maintaining the mixture below said pH at a temperature of between 10° C. and 150° C.; and/or (b) contacting the complex mixture with a carbon dioxide source; and (c) recovering the cephalosporanic acid compound of formula (I) from the mixture obtained after steps (a) and/or (b).

FIELD OF THE INVENTION

[0001] The present invention relates to a process for the preparation ofcephalosporins and cephalosporin derivatives. More in particular, thepresent invention relates to the recovery of cephalosporins andderivatives thereof from complex mixtures of cephalosporins and otherbeta-lactam compounds. The invention is also concerned with recovery ofdeacylated cephalosporins from mixtures of beta-lactam compounds andside chains, such as those obtainable by enzymatic side-chain removal.

BACKGROUND OF THE INVENTION

[0002] Semi-synthetic routes to prepare cephalosporins mostly start fromfermentation products such as penicillin G, penicillin V andCephalosporin C, which are converted to the corresponding β-lactamnuclei, for instance in a manner as is disclosed in K. Matsumoto,Bioprocess. Techn., 16, (1993), 67-88, J. G. Shewale & H. Sivaraman,Process Biochemistry, August 1989, 146-154, T. A. Savidge, Biotechnologyof Industrial Antibiotics (Ed. E. J. Vandamme) Marcel Dekker, New York,1984, or J. G. Shewale et al., Process Biochemistry International, June1990, 97-103. The obtained β-lactam nuclei are subsequently converted tothe desired antibiotic by coupling to a suitable side chain, as has beendescribed in inter alia EP 0 339 751, JP-A-53005185 and CH-A-640 240. Bymaking different combinations of side chains and β-lactam nuclei, avariety of penicillin and cephalosporin antibiotics may be obtained.

[0003] 7-Amino desacetoxy cephalosporanic acid (7-ADCA) and7-aminocephalosporonic acid (7-ACA) are known to be the most importantintermediates for the production of antibiotics used in thepharmaceutical industry.

[0004] 7-ADCA is for example obtained by chemical or enzymatic cleavage(deacylation) of phenylacetyldesacetoxy cephalosporanic acid yielding7-amino desacetoxy cephalosporanic acid and phenyl acetic acid.

[0005] Phenylacetyldesacetoxy cephalosporanic acid is normally producedby chemical treatment of penicillin G sulfoxide, which is formed frompenicillin G. In this production process a large amount of chemicals arerequired to ensure that the desired reaction take place. This is bothexpensive and places a heavy burden on waste management. Moreover, thetotal yield of the process, is not very high.

[0006] To overcome some of the drawbacks of the chemical process afermentative process has been disclosed for the production of 7-ADCA,7-amino desacetyl cephalosporanic acid (7-ADAC) and 7-ACA, involvingfermentative production of N-substituted β-lactams, such asadipyl-7-ADCA, adipyl-7-ADAC or adipyl-7-ACA by a recombinantPenicillium chrysogenum strain capable of expressing adesacetoxycephalosporanic acid synthetase (DAOCS) also known as“expandase” from a transgene (EP 0 532 341, EP 0 540 210, WO 93/08287,WO 95/04148). The expandase takes care of the expansion of the5-membered ring of certain N-acylated penicillanic acids, therebyyielding the corresponding N-acylated desacetoxycephalosporanic acids.

[0007] In order to yield the economically most important non-acylatedcephalosporins, such as 7-ADCA, 7-ADAC and 7-ACA, the acyl groups areenzymatically removed with a suitable acylase.

[0008] Known processes for recovering chemically or enzymaticallyproduced penicillanic and cephalosporanic acids are not effective forthe recovery of the N-substituted β-lactam intermediates and deacylatedamino-β-lactams. The main problem with the recovery of thefermentatively produced cephalosporin compounds mentioned above is thecomplexity of the broth, or culture filtrate. The broth usuallycomprises various penicillanic acids, such asalpha-aminoadipyl-6-penicillanic acid,alpha-hydroxyadipyl-6-penicillanic acid, 6-aminopenicillanic acid(6-APA), various cephalosporanic acids including alpha-aminoadipyl- andhydroxyadipyl-7-ADCA and a lot of proteinaceous material. Known recoveryprocedures do not give an acceptable quality of the cephalosporanic acidproduct in terms of purity. In deacylation this leads to problems interms of reduced enzyme half-life, slower bioconversion rate and moreexpenses in the recovery after bioconversion and/or unacceptablecontaminant levels. Moreover, after deacylation, such impurities preventor at least hamper the recovery of the desired deacylated cephalosporincompound of the desired specifications.

SUMMARY OF THE INVENTION

[0009] The invention provides for a method for the recovery of acephalosporanic acid compound of the general formula (I):

[0010] wherein

[0011] R₀ is hydrogen or C₁₋₃ alkoxy;

[0012] Y is CH₂, oxygen, sulphur, or an oxidised form of sulphur;

[0013] R₁ is any of the groups selected from the group consisting of

[0014] hydrogen,

[0015] hydroxy,

[0016] halogen,

[0017] saturated or unsaturated, straight or branched alkyl (1-5 carbonatoms; optionally replaced by one or more heteroatoms), optionallysubstituted with hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), oracyl;

[0018] alkoxy (1-3 carbon atoms; optionally replaced by one or moreheteroatoms), optionally substituted with hydroxy or halogen; or

[0019] cycloalkyl (3-8 carbon atoms) optionally substituted withhydroxy, halogen, amino;

[0020] aryl;

[0021] heteroaryl; and

[0022] R₂ is selected from the group consisting of adipyl(1,4-dicarboxybutane), succinyl, glutaryl, adipyl, pimelyl, suberyl,2-(carboxyethylthio)acetyl, 3-(carboxy-ethylthio)propionyl, higher alkylsaturated and higher alkyl unsaturated dicarboxylic acids,

[0023] from a complex mixture comprising in addition to the compound ofthe general formula 6-aminopenicillanic acid (6-APA) and optionally oneor more N-substituted β-lactam compounds,

[0024] comprising the steps of:

[0025] (a) acidifying the complex mixture to a pH below 6.5 andmaintaining the mixture below said pH at a temperature of between 10° C.and 150° C.; and/or

[0026] (b) contacting the complex mixture with a carbon dioxide source;and

[0027] (c) recovering the cephalosporanic acid compound of the formula(I) from the mixture obtained after steps (a) and/or (b).

[0028] Preferably in step (a) the temperature is kept between about 50°C. and about 130° C., preferably between 70 and 120° C., for between 10seconds and about 1 week and the pH is kept at or below pH 4.5.According to a preferred method the compound of formula (I) has beenproduced by fermentation of a micro-organism capable thereof, thecomplex mixture being a broth, a culture filtrate or any culture liquidderivable from the broth after fermentation.

[0029] Preferred compounds of the general formula (I) are selected fromthe group consisting of adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA.

[0030] According to another aspect of the invention step (c) isperformed by subjecting the mixture obtained after steps (a) and/or (b)to chromatography, preferably adsorption chromatography, more preferablyHydrophobic Interaction Chromatography.

[0031] According to another aspect of the invention the use ofchromatography in a process of recovering a cephalosporin compoundaccording to formula (I) is provided, preferably by adsorptionchromatography, more preferably Hydrophobic Interaction Chromatography,still more preferably using Simulated Moving Bed technology.

[0032] According to yet another aspect of the invention a method isprovided for making a compound of formula (II):

[0033] wherein

[0034] R₀ is hydrogen or C₁₋₃ alkoxy;

[0035] Y is CH₂, oxygen, sulphur, or an oxidised form of sulphur;

[0036] R₁ is any of the groups selected from the group consisting of

[0037] hydrogen,

[0038] hydroxy,

[0039] halogen,

[0040] saturated or unsaturated, straight or branched alkyl (1-5 carbonatoms; optionally replaced by one or more heteroatoms), optionallysubstituted with hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), oracyl;

[0041] alkoxy (1-3 carbon atoms; optionally replaced by one or moreheteroatoms), optionally substituted with hydroxy or halogen; or

[0042] cycloalkyl (3-8 carbon atoms) optionally substituted withhydroxy, halogen, amino;

[0043] aryl;

[0044] heteroaryl;

[0045] comprising the steps of making a compound according to formula(I) wherein R₀, Y and R₁ are as above and R₂ is selected from the groupconsisting of adipyl (1,4-dicarboxybutane), succinyl, glutaryl, adipyl,pimelyl, suberyl, 2-(carboxyethylthio)acetyl,3-(carboxyethylthio)-propionyl, higher alkyl saturated and higher alkylunsaturated dicarboxylic acids;

[0046] deacylating the compound of formula (I) to obtain a conversionsolution which comprises a compound according to formula (II).

[0047] The conversion solution preferably further comprises the cleavedside chain designated R₂.

[0048] According to a preferred embodiment, the process comprises thefurther step of recovering the compound of formula (II) from thesolution by crystallisation, preferably preceded and/or followed (aftersolubilisation of the crude crystals i.e. by crystallisation) bytreatment of the solution with an agent selected such as activatedcarbon or an adsorber resin. According to another aspect of theinvention during or before crystallisation and/or recrystallisation asolvent such as methanol, ethanol, (iso)propanol, isobutanol, n-butanol,or acetone or a combination of any of the mentioned agents is added.Preferred adsorber resins are selected from XAD16 (CAS No. 102419-63-8),XAD1600 (CAS No. 153796-66-8) and HP20 (CAS No. 55353-13-4). Preferredaccording to the invention is a method wherein the 6-aminopenicillanicacid (6-APA) level is 10 ppm or less with respect to the compound offormula (II). According to another aspect, a process is provided whereinfollowing the deacylation the solution is treated to remove, at leastpartially, the cleaved side chain represented by R₂. This step may beperformed, or repeated, after crystallisation and solubilisation (i.e.recrystallisation) of the compound of formula (II). Also removal of thecleaved side-chain may be carried out on the mother liquor obtainedafter crystallisation or recrystallisation.

[0049] Thus, a process is provided wherein said treatment to remove, atleast partially, the cleaved side chain is followed by solubilisation ofthe crude crystals and recrystallisation of the compound of formula(II).

[0050] Preferably said treatment to remove the cleaved side chaincomprises subjecting the conversion solution, or the mother liquor, orboth, to membrane filtration at a pH below 5, preferably below 4, morepreferably near or below 3. Accordingly, the use is provided of membranefiltration to remove a dicarboxylic acid from a mixture comprising thedicarboxylic acid and a β-lactam antibiotic. The mixture is preferably amother liquid obtained after crystallisation of a compound of theformula (II) or the mixture obtained after deacylation of the compoundof formula (I). Membrane filtration takes preferably place at a pH ofabout 5 or less, preferably at pH 4 or less, yet more preferably bynanofiltration at or below pH 3.

[0051] According to another aspect of the invention, a process isprovided wherein the side chain R₂ is, at least partially, removed fromthe conversion mixture by crystallisation and/or recrystallisation.

[0052] According to still another aspect of the invention, a process isprovided wherein the side chain R₂ is, at least partially, removed fromthe conversion mixture by acidifying the mixture to a pH lower than 3and next contacting this mixture with an organic solvent, for instanceamyl acetate, butyl actetate, ethyl acetate, methyl isobutyl ketone,cyclohexanone, isobutanol or n-butanol.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The invention pertains to a method for the recovery of acephalosporanic acid compound of the general formula (I):

[0054] wherein

[0055] R₀ is hydrogen or C₁₋₃ alkoxy;

[0056] Y is CH₂, oxygen, sulphur, or an oxidised form of sulphur;

[0057] R₁ is any of the groups selected from the group consisting of

[0058] hydrogen,

[0059] hydroxy,

[0060] halogen,

[0061] saturated or unsaturated, straight or branched alkyl (1-5 carbonatoms; optionally replaced by one or more heteroatoms), optionallysubstituted with hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), oracyl;

[0062] alkoxy (1-3 carbon atoms; optionally replaced by one or moreheteroatoms), optionally substituted with hydroxy or halogen; or

[0063] cycloalkyl (3-8 carbon atoms) optionally substituted withhydroxy, halogen, amino;

[0064] aryl;

[0065] heteroaryl; and

[0066] R₂ is selected from the group consisting of adipyl(1,4-dicarboxybutane), succinyl, glutaryl, adipyl, pimelyl, suberyl,2-(carboxyethylthio)acetyl, 3-(carboxy-ethylthio)propionyl, higher alkylsaturated and higher alkyl unsaturated dicarboxylic acids,

[0067] from a complex mixture comprising in addition to the compound ofthe general formula 6-aminopenicillanic acid (6-APA) and optionally oneor more N-substituted penicillanic acid compounds,

[0068] comprising the steps of:

[0069] (a) acidifying the complex mixture to a pH below 6.5 andmaintaining the mixture below said pH at a temperature of between 10° C.and 150° C.; and/or

[0070] (b) contacting the complex mixture with a carbon dioxide source;and

[0071] (c) recovering the cephalosporanic acid compound of the formulafrom the mixture obtained after steps (a) and/or (b). The inventionrelates further to a process for the preparation of cephalosporinshaving the general formula (II):

[0072] wherein

[0073] R₀ is hydrogen or C₁₋₃ alkoxy;

[0074] Y is CH₂, oxygen, sulphur, or an oxidised form of sulphur;

[0075] R₁ is any of the groups selected from the group consisting of

[0076] hydrogen,

[0077] hydroxy,

[0078] halogen,

[0079] saturated or unsaturated, straight or branched alkyl (1-5 carbonatoms; optionally replaced by one or more heteroatoms), optionallysubstituted with hydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), oracyl;

[0080] alkoxy (1-3 carbon atoms; optionally replaced by one or moreheteroatoms), optionally substituted with hydroxy or halogen; or

[0081] cycloalkyl (3-8 carbon atoms) optionally substituted withhydroxy, halogen, amino;

[0082] aryl;

[0083] heteroaryl.

[0084] The compound according to formula (I) may be produced by anyseries of steps which yield a complex mixture as defined herein, fromwhich the recovery of the compound according to formula (I) isaccomplished. For the purposes of the specification and claims, acomplex mixture is defined as a mixture comprising a N-substitutedcephalosporin compound and substituted or unsubstituted β-lactamcompounds.

[0085] The compound of formula (II) is obtained by the following seriesof steps:

[0086] (a) recovering, preferably purifying, the compound of formula(I);

[0087] (b) deacylating the preferably purified compound of formula (I)to obtain a solution comprising the compound of formula (II) (theconversion solution); and

[0088] (c) recovering, preferably purifying the compound of formula(II).

[0089] One of the obstacles of producing N-substituted cephalosporanicacid fermentatively is the presence of unwanted contaminating β-lactamcomponents, for instance N-substituted 6-amino penicillanic acid.According to one embodiment of the invention, it has been found thatthese contaminations can be remarkably reduced by incubating the broth,the filtrate of the broth or a liquid derived from the broth using anybiomass separation technique, under acidified conditions, preferablyaccompanied with an elevated temperature. The broth is acidified down toa pH lower than 6.5, preferable lower than 4.5, using at least one knownacid, for instance sulphuric acid, hydrochloric acid or nitric acid or acombination thereof. Operating temperature is in the range of 20 to 150°C., preferably at 70 to 120° C. Residence time at these conditions is inthe range of a few seconds (at 150° C.), or several days (at 20° C.),preferably 10 seconds to 60 minutes. The pH/temperature treatment ispreferably carried out for a period which provides for an N-substituted6-APA reduction of a factor 100, preferably 1000, more preferably1,000,000 with respect to the compound of formula (II). This step can becarried out either before or after biomass separation and can beperformed batch wise or continuously.

[0090] According to another embodiment of the invention contaminatingpenicillin components, for instance N-substituted 6-APA, are remarkablyreduced by contacting the broth, the filtrate of the broth, the eluate,the conversion solution or the dissolved contaminated cephalosporinaccording to formula (I), typically at pH 5 to 7, with carbon dioxide.Carbon dioxide can be added to the solution in any suitable way, such assolid or gaseous form or as solution of carbonate ions. The solution iscontacted with the CO₂ source at a temperature of 10 to 60° C.,preferably 20 to 40° C., where said solution is saturated with molecularCO₂ for 4 to 10 hours. After reduction of the penicillin components,purification of the cephalosporins, according to formula 1 can beobtained as mentioned earlier.

[0091] The complex mixture as defined herein may have any origin, but ispreferably a culture broth or a culture filtrate obtained afterfermenting under conditions giving rise to production, a micro-organismcapable of producing an 7-N-acylated version of the compound of thegeneral formula (I), wherein the acyl-group may be any acyl-group whichsupports the ring-expanding enzyme (desacetoxycephalosporinsynthetase—DAOCS—or a bifunctional expandase/hydroxylase occasionallyreferred to as desacetylcephalosporin synthetase DACS) in thecephalosporin biosynthetic pathway. Bioprocesses for producing 7-Nacyl-substituted compounds according to formula (I) in vivo aredisclosed in WO 93/05158 (adipyl-7-ADCA); WO 93/08287 (adipyl-7-ADAC andadipyl-7-ACA), WO 95/04148 (2-(carboxyethylthio)acetyl-7-ADCA), WO95/04149 (3-(carboxyethylthio)propionyl-7-ADCA) and higher alkylsaturated or unsaturated dicarboxylic acids. The relevant parts of thesePCT-applications are herein incorporated by reference. Preferred acylgrouts are dicarboxylic acid groups in general, such as adipyl(1,4-dicarboxybutane), 2-(carboxyethylthio)acetyl,3-(carboxyethylthio)propionyl, muconic acid and the like. Suitable hostorganisms include but are not limited to Penicillium chrysogenum andAcremonium chrysogenum. Suitable sources of expandases, includingbifunctional expandase/hydroxylases include but are not limited toStreptomyces clavuligerus and Acremonium chrysogenum. Methods fortransformation, selection of transformed cells and expression regulatingelements for filamentous fungi, which may be used to genetically modifyhost cells, are well known in the art of recombinant DNA technology ofβ-lactam producing (filamentous) fungi.

[0092] Preferably, the broth is subjected first to biomass separationsuch as filtration by any suitable means, such as membrane filtration,vacuum filtration, ultrafiltration or a combination thereof, prior toacidification and the optional temperature increase. Any other means ofbiomass separation is suitable as well.

[0093] After the pH-lowering step and the optional temperature step, therecovered compound according to the formula (I) is preferably subjectedto further purification to remove, at least partially, unwanted β-lactamcomponents, especially unwanted N-substituted cephalosporins andpenicillins. The further purification may be carried out by extractionusing an organic solvent. In the case of extraction, it is found to beadvantageous to wash the extract, back extract the N-substitutedcephalosporin from the organic phase to an aqueous phase and strippingthe aqueous phase. The extracting organic solvent may be selected fromamyl acetate, butyl acetate, ethyl acetate, methyl isobutyl ketone,cyclohexanone, iso-butanol or n-butanol, and the like. A preferredpurification step in the process is the usage of chromatography for thepurification of N-substituted cephalosporin, rather than extractionusing organic solvents. The advantage of chromatography is in theabsence of solvents, which cause waste problems and problems ofcontainment, as well as improved purity of the final product. Preferredis ion exchange chromatography or adsorption chromatography, morepreferably Hydrophobic Interaction Chromatography. The filtrate issubjected to chromatography using an adsorbent. An adsorbent includesactivated carbon, e.g. Norit CG-1 or Cecarbon GAC 40; or an adsorberresin, such as styrene-divinylbenzene copolymerisates, for exampleDianion HP 20 (CAS No. 55353-13-4), Dianion HP 21 (CAS No. 92529-04-9),Dianion SP 207 (CAS No. 98225-81-1) or Dianion SP825, from MitsubishiKasei Corporation or Amberlite XAD 1180 (CAS No. 97396-56-0), AmberliteXAD 1600 (CAS No. 153796-66-8) or Amberlite XAD 16 (CAS No. 102419-63-8)from Rohm and Haas or Amberchrom CG 161 (CAS No. 131688-63-6) fromTosoHaas; preferably XAD 16 or XAD 1600.

[0094] Before adsorbing the N-substituted cephalosporin the complexmixture is adjusted to a pH of 1.0 to 5.0, preferably 2.5 to 3.5, by themeans of one or more known acids, for instance sulphuric acid,hydrochloric acid or nitric acid or a combination thereof. Operatingtemperature is the range of 0 to 50° C., preferably at 5 to 25° C.Operating pressure is in the range of 0 to 1.0 MPa overpressure.

[0095] Unwanted β-lactam components, especially unwanted N-substitutedcephalosporins, such as alpha-aminoadipylcephalosporanic acids, alsoadsorb on the adsorbent but are displaced by the wanted N-substitutedcephalosporin.

[0096] After adsorbing, washing with water is applied to remove unwantedβ-lactam components from the void volume between the adsorbent and todesorb weakly bound unwanted β-lactam components from the adsorbent. Thewater can be acidified down to a pH of 1.0 by the means of one or moreknown acids, for instance sulphuric acid, hydrochloric acid or nitricacid or a combination thereof. To increase the osmotic pressure, saltsmay be added to the water. Operating temperature is the range of 0 to50° C., preferably at 20 to 40° C. Operating pressure is in the range of0 to 1.0 MPa overpressure.

[0097] Elution may be carried out with a suitable buffer, such asacetate, phosphate, carbonate, bicarbonate or adipate but also dilutedorganic solvents (e.g. acetone, isopropanol) or diluted bases (e.g.ammonium, caustic) can be used. Operating temperature is the range of 0to 80° C., preferably at 10 to 40° C. Operating pressure is in the rangeof 0 to 1.0 MPa overpressure.

[0098] Regeneration of the adsorbent can be done by any common appliedmethod, such as with dilute bases, dilute acids, or with water misciblesolvents (such as acetone, methanol, ethanol or iso-propanol), or acombination thereof. Optionally heating up to 100° C. may be performed.

[0099] The regeneration liquids can be removed by washing with water.The water can be acidified down to a pH of 1.0 by the means of one ormore known acids, for instance sulphuric acid, hydrochloric acid ornitric acid or a combination thereof.

[0100] The chromatography step can be performed in several types ofequipment, such as in a single column but also the simulated moving bedtechnology can be applied. For this simulated moving bed technologyseveral types of equipment are available, such as the ADSEP system fromU.S. Filter, the ISEP/CSEP-system from Advanced Separation Technology,the ‘merry-go-around’-system from e.g. Applexion or the SORBEX-systemfrom Universal Oil Products Company (UOP).

[0101] Alternatively, the buffer can be removed from the eluate by meansof nanofiltration. The characteristics of the membrane in this membranefiltration show a high retention for the wanted N-substitutedcephalosporin and a low retention for the buffer.

[0102] Optionally a concentration step is applied by any means ofsuitable concentration such as vacuum evaporation, reversed osmosis,nanofiltration, or narofiltration after chromatography or extraction.

[0103] The recovered N-acylated compound is subsequently subjected todeacylation using any suitable method known in the art. A preferredmethod is enzymatic deacylation using a suitable dicarboxylate acylase.Numerous suitable acylases, wild-type or mutated, are known in the artincluding but not limited to those from Bacillus (EP 0 525 861; EP 0 405846) Pseudomonas (EP 0 482 844; EP 0 525 861; EP 0 475 652; EP 0663445), Achromobacter (EP 0 525 861), Alcaligenes faecalis (EP 0 638 649),Acinetobacter (EP 0 469 919), Arthrobacter (EP 0 283 218), Escherichiacoli (U.S. Pat. No. 3,945,888), Kluyvera citrophila, Proteus rettgeri(U.S. Pat. No. 3,915,798) and the like. The dicarboxylate acylase ispreferably from Pseudomonas SE83 or SY-77. Optionally, the acylase maybe a mutated form, as disclosed in WO 91/16435, WO 97/20053, WO 97/40175to increase or alter the affinity towards the substrate. Another way ofdeacylating the N-acylated cephalosporin compound according to theinvention is by way of contacting the substrate with a micro-organismcapable of producing the acylase, as disclosed in U.S. Pat. No.5,677,141.

[0104] The acylase may be immobilised (U.S. Pat. No. 3,930,949), eitheron membranes (EP 0 243 404) or free flowing carriers such asglutaraldehyde based carriers or aza-lacton polymers (EP 0 730 035),using techniques as such well known in the art. Non-immobilised acylaseis also contemplated, using membranes to separate the reaction mixture(retentate) from the product (permeate), such as disclosed in U.S. Pat.No. 5,521,068. The process may be batch-wise or (semi-) continuous, thisis all well known and not crucial with respect to the invention. Theenzymatic deacylation reaction is usually carried out in a stirred tankreactor with or without, preferably inert, sieve plates, to easilyseparate the immobilised enzyme from the reaction product. The pH isusually regulated during the reaction to compensate for the pH change asa result of the (dicarboxylic) side-chain removal by any type of basesuch as ammonium, caustic, carbonate, bicarbonate. The pH can beregulated in the reactor and/or in a circulating loop over the reactor.Other parameters may also be regulated, such as temperature, deacylatedproduct or side-chain concentration, and the like, taking account of theeffect of such parameters on the reaction rate and/or the equilibrium.

[0105] Additional stabilising agents can be added before and/or duringdeacylation, such as sulphite (S₂O₅ ²⁻, HSO₃ ⁻, SO₃ ²⁻), EDTA,dithiotreitol (DTT).

[0106] Usually, the deacylated cephalosporin compound of the generalformula (I) is subsequently recovered using any suitable combination ofsteps. Optionally a concentration step can be applied by any means suchas vacuum evaporation, reversed osmosis, nanofiltration, ornarofiltration before crystallisation. Optionally a water misciblesolvent can be added. Optionally, before crystallisation the solutioncan be purified by treating with activated carbon or an adsorber resin.Optionally, before crystallisation the side chain can be removed,characterised by acidifying the aqueous phase, extracting the side chainto an extracting organic solvent and separating the phases. Theextracting organic solvent may be selected from amyl acetate, butylacetate, ethyl acetate, methyl isobutyl ketone, cyclohexanone,iso-butanol, n-butanol, and the like.

[0107] The product can be crystallised from the resulting aqueous phasein several ways. The most preferred mode of operation is neutralisingthe aqueous solution and subsequently lowering the pH in 1 to 6 stepsdown to a pH 3 to 5 using one or more known acids such as H₂SO₄, HCl,HNO₃, or a combination thereof. This is preferably carried out incontinuous mode using an interconnected set of 1 to 6 continuouslyoperated crystallisers in series. Also batch crystallisation,semi-continuous crystallisation or concordance crystallisation can beapplied. It is possible to perform the crystallisation directly in thesame way as above, without the first neutralisation. According to oneembodiment of this invention it has been found that a water misciblesolvent, such as methanol, ethanol, iso-propanol, n-butanol, acetone andthe like, can be added to improve the quality of the cephalosporinaccording to formula (II) Optionally, before crystallisation thesolution can be treated by activated carbon or by an adsorbent resin inorder to improve the quality of the compound according to formula (II).

[0108] It has been found that the quality of the cephalosporin accordingto formula (II) can be further improved by recrystallisation, optionallyafter treatment with adsorber resins, active coal and/or ethanol and/oracetate. This is characterised by dissolving the cephalosporin accordingto formula (II) at a pH in the range of 0.5 to 10.0, preferably between7.5 to 8.5 and crystallisation of the product. The product can becrystallised in several ways. The most preferred mode of operation islowering of the pH in 1 to 6 steps down to a pH 3 to 5 using one or moreknown acids, such as H₂SO₄, HCl, HNO₃, or a combination thereof. Thiscan be carried out in continuous mode using an interconnected set of 1to 6 continuously operated crystallisers in series. Also batchcrystallisation, semi-continuous crystallisation or concordancecrystallisation can be applied. According to one embodiment of thisinvention it has been found that a water miscible solvent, such asmethanol, ethanol, (iso)propanol, acetone, iso-butanol and n-butanol,can be added to improve the quality of the cephalosporin according toformula (II).

[0109] It has been found further, that the quality of the cephalosporinaccording to formula (II) can be improved by treating the conversionsolution and/or the solution of the dissolved cephalosporin according toformula (II) with an adsorbent. An adsorbent includes activated carbon,e.g. Norit Ultra SX; or an adsorber resin, such asstyrene-divinylbenzene copolymerisates, for example Dianion HP 20 (CASNo. 55353-13-4), Dianion HP 21 (CAS No. 92529-04-9) or Dianion SP 207(CAS No. 98225-81-1) from Mitsubishi Kasei Corporation or Amberlite XAD1180 (CAS No. 97396-56-0), Amberlite XAD 1600 (CAS No. 153796-66-8) orAmberlite XAD 16 (CAS No. 102419-63-8) from Rohm and Haas or AmberchromCG 161 (CAS No. 131688-63-6) from TosoHaas; preferably XAD 16, XAD 1600or HP20.

[0110] The crystals are isolated by filtration or centrifugation anddried in a conventional continuous or batch dryer. The crystals can bemilled by any type of mill, such as ball mill, jet mill and the like.

[0111] Optionally a water miscible solvent can be added during thecrystallisation. After dissolving, the solution can be treated withactivated carbon or an adsorber resin.

[0112] This procedure will gave a better overall yield and productquality than the currently known process, mentioned before.

[0113] According to another aspect of the invention, a method isprovided for removing and recovering adipic acid from the conversionsolution or mother liquid (the liquid obtained after crystallisation ofthe compound according to the formula (II)). It is found, that adipicacid can advantageously separated using membrane filtration at low pH,such as below pH 5, preferably below pH 4, more preferably below at ornear pH 3. Preferred according to the invention is an embodiment whereinfiltration is carried out by reversed osmosis.

[0114] In addition to saving raw materials, the advantage of doing soresides in the purity and/or yield upon crystallisation of theso-treated solution.

[0115] The invention is further illustrated by the followingnon-limiting examples.

[0116] Experimental

[0117] A fermentation broth comprising adipyl-7-ADCA as a complexmixture, comprising inter alia 6-APA, adipyl-6-APA andalpha-amino-adipyl-7-cephalosporanic acid as undesired contaminants, isobtained by fermenting a Penicillium chrysogenum strain transformed withan expandase (desacetoxycephalosporin C synthetase) from Streptomycesclavuligerus, as described in International patent application WO93/05158, published on Mar. 18, 1993.

[0118] The transformed Penicillium strain was cultured as described inExample 1 of WO 93/05158, incorporated by reference herein.

[0119] After 5 to 7 days of fermentation, the broth was taken forrecovery experiments.

[0120] This complex mixture can also be simulated by making an aqueousmixture of 6-aminopenicillanic acid, adipyl-6-aminopenicillanic acid,alpha-aminoadipyl-6-amino-penicillanic acid,adipyl-7-aminodesacetoxycephalosporanic acid, andalpha-aminoadipyl-7-cephalosporanic acid.

EXAMPLE 1 pH/Heat-Treatment

[0121] This example shows the advantages of a pH-treatment, preferably acombined pH-plus increased temperature treatment, on the removal ofunwanted β-lactam components, from complex mixtures.

[0122] Broth from a fermentation of Penicillium chrysogenum (seeexperimental), containing a complex mixture of adipyl-7-ADCA andpenicillanic acid and cephalosporanic acid contaminants is filtrated.The concentrate is washed with process water until the total volume ofthe combined filtrates was approx. 2 times the initial broth volume.

[0123] The following experiments have been carried out:

[0124] A. Part of the filtrate is acidified to pH=3.5; heated up to 70°C. and after 30 minutes cooled to 40° C.;

[0125] B. Part of the permeate is acidified to pH=2.7; heated up to 110°C. and after 4 minutes cooled to 25° C.; or

[0126] C. Part of the permeate is acidified to pH=3.0 and not furthertreated.

[0127] The pre-treated solutions are then subjected to the followingtreatments to obtain a compound according to formula (II); 7-ADCA.

[0128] Adsorption Chromatography

[0129] The three solutions (A to C) were subjected to filtration over aSeitz K100 filter, whereafter the solution was pumped at a pH of 3.0over a column filled with 1.6 liter of XAD-1600 resin; next the resinwas washed with 4.8 liter water, and eluted with 0.2 Mbicarbonate-solution. The first eluate fraction (1.1 liter) is taken outand discarded. The second fraction (3.2 liter) is collected andanalysed. The resin is purified by washing with caustic and acetone, andconditioned again with acidified water.

[0130] Concentrating

[0131] The eluate was concentrated at 20 to 30° C. vacuum (5-10 mm Hg)till a concentration of 40 grams adipyl-7-ADCA per liter was obtained.

[0132] Enzymatic Deacylation

[0133] Subsequently, the adipyl-7-ADCA is treated with acylase asfollows. To 1 liter of eluate, 1 gram of sodium metabisulfite, 20 mMEDTA and 100 g immobilised acylase (comprising Pseudomonas SE83dicarboxylate acylase) was added. At 30° C., the solution was stirredfor two hours. The pH was held at 8.5 with 4 N sodium hydroxide. Theimmobilised acylase and the liquid were separated with a glass sinteredfilter.

[0134] Crystallisation of 7-ADCA

[0135] The 7-amino desacetoxy cephalosporanic acid (7-ADCA) wasprecipitated by lowering the pH to 3.6, under stirring, at a temperatureof 30° C.; in 45 minutes the pH of the solution was lowered to 3.6 with6 N sulphuric acid. After cooling to 20° C., the crystals were isolatedon a glass sintered filter, washed with water and dried at 35° C.

[0136] Resolving 7-ADCA Crystals

[0137] The 7-ADCA was dissolved with the aid of ammonia. To that end 15grams of 7′-ADCA was suspended in 255 ml water. The 7-ADCA was dissolvedwith the aid of 4 N ammonium hydroxide at a pH of 7.5-8.5. Afterfiltration over a glass sintered filter, water was added to obtain 300ml of solution.

[0138] Treatment with Adsorber Resin

[0139] The solution was treated with adsorber resin. In 45 minutes thesolution was pumped over 15 ml of XAD1600. Subsequently, 75 ml of waterwas pumped over the resin to obtain 375 ml of solution.

[0140] Recrystallisation

[0141] The 7-ADCA was precipitated by lowering the pH to 3.6 understirring, at a temperature of 30° C.; in 45 minutes the pH was loweredto 3.6 with 6 N sulphuric acid. After cooling to 20° C., the crystalswere isolated on a glass sintered filter, washed with water and dried at35° C.

[0142] The 7-ADCA so produced shows good results in terms of 6-APAreduction. (6-APA ratio is with respect to 7-ADCA) TABLE 1a Results ofexperiment 1A, 1B and 1C. 6-amino penicillanic acid content Experiment(ppm) 1A <10 1B <10 1C 950

[0143] Clearly, the pH/Temperature treatment reduces the level of6-aminopenicillanic acid contamination of the adipyl-7-ADCA preparation.ship between pH, Temperature and Time ermined for a fixed reduction of6-id of 10⁻⁶ (Table 1b). TABLE 1b 6-APA Temp. Time Time Time reductionpH (C.) (s) (min) (h) 10⁻⁶ 3 25 35050 584 9.74 10⁻⁶ 3 50 3057 50.9 0.8510⁻⁶ 3 75 378 6.3 0.11 10⁻⁶ 3 100 62 1.0 0.02 10⁻⁶ 4 25 148857 248141.35 10⁻⁶ 4 50 12982 216.4 3.61 10⁻⁶ 4 75 1607 26.8 0.45 10⁻⁶ 4 100 2634.4 0.07

EXAMPLE 2 Adsorption Chromatography

[0144] This example shows the effect of (a) the degree of loading of thecolumn when adsorption chromatography is used (2A to 2D), (b) the effectof washing the column with different amounts of water prior to elution(2E to 2G), (c) the effect of the pH of the feed on the purification ofadipyl-7-ADCA (2H to 2J). The embodiment where adsorption chromatographyis carried out in a Simulated Moving bed mode is given as Experiment 2K.

[0145] The broth is pre-treated as described in Example 1A. Theadipyl-7-amino-desacetoxy cephalosporanic acid was subsequently purifiedby adsorption chromatography by pumping the solution over a columnfilled with 1.6 liter of XAD-1600 resin, washed with different amountsof water (2A to 2D and 2H to 2K: 4.8 liter; 2E to 2F: see Table 2b), andeluted with 0.2 M bicarbonate-solution. The first eluate fraction (1.1liter) is taken out and discarded. The second fraction (3.2 liter) iscollected and analysed. The resin is purified by washing with causticand acetone, and conditioned again with acidified water. Several changesin process conditions were applied (see table 2). Reduction iscalculated as:(comp-i_(feed)/comp-l_(feed))/(comp-i_(eluate)/comp-l_(eluate)). TABLE2a Results of experiment 2 Feed Eluate (g) Reduction comp 1 comp 2 comp3 comp 1 comp 2 comp 3 (−) comp 4 Exp (g) (g) (g) (g) (g) (g) comp 2comp 3 (ppm) 2A 34 3.3 9.3 30 3.1 5.9 1 1 2B 80 5.9 18.5 70 0.5 0.09 10173 <6 2C 127 10.5 32.8 76 0.3 0.07 24 301 19 2D 255 18.2 59.4 67 0.10.03 38 501 30

[0146] These results clearly show the positive effect of overloading thecolumn on the reduction of compounds 2 and 3 in the eluate. TABLE 2bResults of experiment 2 Feed Eluate Reduction comp 1 comp 2 comp 3 Washcomp 1 comp 2 comp 3 (−) Exp (g) (g) (g) (1) (g) (g) (g) comp 2 comp 32E 85 6.6 14.0 1.6 81 2.1 1.2 3 11 2F 74 6.4 12.6 4.8 71 1.1 0.13 6 942G 75 5.8 13.0 7.3 68 0.5 0.1 12 122

[0147] The example 2b shows the positive effect of extended washing,prior to elution with sodium bicarbonate, on the reduction of undesiredcephalosporin compounds. TABLE 2c Feed Eluate Reduction comp 1 comp 2comp 3 pH comp 1 comp 2 comp 3 (−) Exp (g) (g) (g) (−) (g) (g) (g) comp2 comp 3 2H 79 8.4 22.2 2.5 83 0.6 0.09 15 248 2I 80 5.9 18.5 2.9 70 0.50.09 10 183 2J 83 8.2 21.7 3.5 62 0.5 0.14 12 118

[0148] The above example shows the effect of the pH at whichcrystallisation was carried out, on the reduction of unwanted 7-Nacylated cephalosporin compounds. H₂SO₄ was used as acid. TABLE 2dResults of experiment 2 (30 litres of resin in an SMB-system wasapplied) Feed Eluate comp 1 comp 2 comp 3 comp 4 comp 1 comp 2 comp 3comp 4 Reduction (−) Exp (kg) (kg) (kg) (kg) (kg) (kg) (kg) (kg) comp 2comp 3 comp 4 2K 1.46 0.08 0.20 0.15 1.38 0.01 0.01 0.02 7 18 7

[0149] This example illustrates the use of adsorption chromatographyperformed according to the so-called Simulated Moving Bed technique, ona kilogram scale. The technique may readily be scaled up further.

[0150] The so treated fractions 2A to 2K were treated with acylase toproduce 7-ADCA as described in Example 1. Excellent conversion resultswere obtained, as illustrated in Example 3.

EXAMPLE 3 Enzymatic Conversion

[0151] This example illustrates the results of enzymatic conversion ofadipyl-7-ADCA to 7-ADCA. The adipyl-7-ADCA was recovered as disclosed inExample 2K (pH-treatment according to Example 1A, adsorptionchromatography was optimised in terms of overloading and washing). Theconversion was carried out as described in Example 1, at the pHindicated in Table 3. Experiment A to E represent different batches.TABLE 3 Product product substrate Substrate stream stream comp 1 comp 2pH comp 1 comp 2 Exp (mmol) (mmol) (−) (mmol) (mmol) A 69.7 2.2 8.5 1.168.6 B 144.2 2.4 8.5 5.9 143.3 C 181.4 2.3 8.5 13.5 174.5 D 113.1 1.7 85.4 108.7 E 113.4 3.1 9 1.2 112.2

[0152] The conversion rate and yields are superior when theadipyl-7-ADCA is pre-treated using the pH/temperature step, as comparedto no treatment. The further purification using chromatography bringsfurther improvement in terms of purity (not shown in the Table).

EXAMPLE 4 Crude Crystallisation

[0153] The broth is pH/heat-treated (Example 1) and enriched inadipyl-7-ADCA by adsorption chromatography as described in Example 2.Subsequently, conversion was carried out as described in Example 1.

[0154] The conversion solution (the solution obtained after deacylation)was concentrated with reversed osmosis to increase the concentration.

[0155] Part of the solution was taken and the 7-ADCA was crystallised bylowering the pH to the pH 3.6, 4 or 5 (see table 4a). TABLE 4a Crudecrystallisation product after comp 1 comp 1 isolation, in in pH washingand product Exp Solution (−) drying (g) (%) A 49.5 3.6 48.9 97.5 B 49.54 48.7 97.4 C 49.5 5 48.2 98

[0156] Crystallisation was satisfactory at all pH tested. In thefollowing experiment the pH was 3.6. The effect of concentrating thesolution is illustrated. TABLE 4b Crude crystallisation product aftercomp 1 in isolation, comp 1 in solution washing and product Exp (g)drying (g) (%) D 15.5 14.8 94.3 E 36.1 35.7 95.3 F 49.5 48.9 97.5

[0157] Clearly, there is an effect of the concentration of 7-ADCA in theconversion solution on purity and yield after crystallisation.

[0158] The following example illustrates the effect of differentadsorbers on product quality (colour in solution and clarity). TABLE 4cCrude crystallisation colour comp 1 comp 1 in in in solution claritysolution product at 425 nm in HCl Exp (g) treatment (%) (−) (EBC) G 25HP20 97.2 0.16 3.6 H 25 HP20 97.8 0.11 2.4 (2 times) I 25 IRA67 97.20.18 6 J 25 IRA67 + HP20 97.7 0.1 0.8 K 25 none 96.3 0.39 7.3

EXAMPLE 5 Treatment of Dissolved 7-Amino Desacetoxy Cephalosporanic Acid

[0159] This example shows the effect on clarity and colour of 7-ADCA,after treating 7-ADCA solution with different adsorber resins, prior tocrystallisation.

[0160] A solution comprising 7-ADCA is made as disclosed in Example 2K(the adsorption chromatography column used is a XAD-1600 resin). TABLE 5Colour in comp 1 solution Clarity dissolved at 425 nm in HC1 Exp (g)treatment (−) (EBC) A 40 — 0.18 4.2 B 40 XAD16 0.05 1.8 C 40 HP20 n.d.0.5 D 40 XAD1600 0.09 0.5 E 25 n.d. 3.6 F 25 +1% EtOH 0.11 0.6 G 50 +3%EtOH 0.18 0.9 H 50 +2% Coal 0.04 n.d. I 50 0.17 1.4 J 50 +5% Coal 0.030.8

EXAMPLE 6 Recovery of Adipyl-7-ADCA Using Extraction with N-Butanol

[0161] Broth comprising adipyl-7-ADCA is treated as described in Example1.

[0162] After acidification, part of the adipyl-ADCA is purified byadsorption chromatography. The solution is pumped over a column filledwith XAD-16 resin, washed with water, and eluted with 0.2 Macetate-solution. The first eluate fraction with low adipyl desacetoxycephalosporanic acid content is taken out and discarded. The secondfraction is collected. The resin is purified by washing with caustic andacetone, and conditioned again with acidified water.

[0163] Part of the adipyl-7-ADCA is purified by means of extraction,followed by washing of the extract, back extraction of the N-substitutedcephalosporin from the organic phase to an aqueous phase and strippingthe aqueous phase; the extracting organic solvent is n-butanol.

[0164] The adipyl-7-ADCA is treated with immobilised acylase to produce7-amino desacetoxy cephalosporanic acid (7-ADCA). Part of the 7-ADCA isisolated by lowering the pH. The adipyl desacetoxy cephalosporanic acidwas dissolved with the aid of caustic. The 7-amino desacetoxycephalosporanic acid was isolated by lowering the pH.

[0165] Part of the 7-amino desacetoxy cephalosporanic acid aqueoussolution is acidified, and the side chain is extracted to an extractingorganic solvent and next the phases are separated; the extractingorganic solvent was n-butanol,

[0166] Finally the crystal cake was filtrated, washed and dried. TABLE 6colour 1 comp 1 in in solution clarity product at 425 nm in HC1 Expdescription (%) (−) (EBC) A extraction/extraction/ 98.3 0.13 2.4crystallisation B chromatography/extraction/ 98.9 0.09 1.5crystallisation C chromatography/ 97.6 0.12 — crystallisation Dchromatography/ 98.7 0.05 1.1 crystallisation/ recrystallisation

[0167] The results using a single extraction are not shown in the table,but the purity is far worse than when chromatography is used, evenwithout recrystallisation. A combination of chromatography andrecrystallisation produces or a combination of chromatography andextraction produces the best results.

EXAMPLE 7 Recovery of Adipic Acid From 7-ADCA Crystallisation MotherLiquors

[0168] 7-ADCA crystallisation mother liquors are obtained as describedin example 4. Adipic acid is determined using HPLC: Aminex HPX-87Hcolumn, 300 mm×7.8 mm, filled with 9 um cation gel (Biorad), eluted at65° C. with a 0.2M solution of H₂SO₄ in water, detection using an RIWaters 410 refractometer. In the examples given below, optimisation isdirected at purity, not at yield.

EXAMPLE 7A Recovery of Adipic Acid Using Acidification

[0169] At 20° C., the pH of 7-ADCA crystallisation mother liquor (250ml, 13.6 g/l adipic acid) was lowered to 0.7 using a 12M solution ofH₂SO₄ in water. After 16 h at 0° C., no crystallisation could bedetected. The pH was raised to 3.4 using a 6M solution of KOH in water.The resulting crystal was recovered by filtration to give 7.7 g ofmaterial which was a mixture of salt and adipic acid which was notfurther analysed.

EXAMPLE 7B Recovery of Adipic Acid Using Acidification and Concentration

[0170] At 20° C., the pH of 7-ADCA crystallisation mother liquor (500ml, 9.4 g/l adipic acid) was lowered to 1.5 using a 12M solution ofH₂SO₄ in water and concentrated under reduced pressure at 40° C. to givea sticky mixture that was isolated by filtration and dried to give 6.9 gadipic acid with a purity of 53% (yield 78%).

EXAMPLE 7C Recovery of Adipic Acid Using Reverse Osmosis with Nanomax 50Membrane

[0171] At 20° C., the pH of 7-ADCA crystallisation mother liquor (500ml, 9.4 to 18.2 g/l adipic acid, see table) was adjusted to the valuementioned in the table using either a 6M solution of KOH in water or a12M solution of H₂SO₄ in water. The resulting solution was subjected toreverse osmosis using a Nanomax 50 membrane from Millipore. With the aidof nitrogen gas, a pressure of 30 bar was applied to give a filtrate anda retentate in which the amount of adipic acid was determined usingHPLC. In most cases, a work-up procedure was applied that consisted ofconcentration under reduced pressure until crystallisation began,followed filtration of the product and drying. TABLE 7 Volume YieldPurity Reten- perme- after after Adipic acid (g/l) tion ate work-upwork-up pH Start Permeate Retentate (%) (ml) (g) (%) 1.5 13.6 11.0 14.826 400 4.4 96 2.0 18.2 13.1 20.8 37 260 2.4 99 3.0 9.4 6.7 8.7 23 3601.9 97 7.2 13.6 5.3 31.9 83 400 no no work-up work-up

EXAMPLE 7D Recovery of Adipic Acid Using Reverse Osmosis with DK U19FMembrane at pH 2.0

[0172] The pH of 7-ADCA crystallisation mother liquor (100 l, containing25.0 g/l adipic acid) was lowered to 2.0 using 2.9 l of a 12M solutionof H₂SO₄ in water. The resulting solution was subjected to reverseosmosis with 57 l water using a DK U19F membrane in a 2.5 m² membranefiltration unit P2-B200 from Hydro Air Research. At a pressure of 30 baran average flux of 8.8 l/m²/h was reached to give the results summarisedin the table. TABLE 8 Concentration (g/l) Retention Component StartPermeate Retentate (%) Adipic acid 25.0 11.6 17.8 35 7-ADCA 0.72 <0.010.72 >99 Adipyl-7-ADCA 1.51 <0.03 1.54 >98

1. A method for the recovery of an N-substituted cephalosporanic acidcompound of the general formula (I):

wherein R₀ is hydrogen or C₁₋₃ alkoxy; Y is CH₂, oxygen, sulphur, or anoxidised form or sulphur; R₁ is any of the groups selected from thegroup consisting of hydrogen, hydroxy, halogen, saturated orunsaturated, straight or branched alkyl (1-5 carbon atoms; optionallyreplaced by one or more heteroatoms), optionally substituted withhydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), or acyl; alkoxy (1-3carbon atoms; optionally replaced by one or more heteroatoms),optionally substituted with hydroxy or halogen; or cycloalkyl (3-8carbon atoms) optionally substituted with hydroxy, halogen, amino; aryl;heteroaryl; and R₂ is selected from the group consisting of adipyl(1,4-dicarboxybutane), succinyl, glutaryl, adipyl, pimelyl, suberyl,2-(carboxyethylthio)acetyl, 3-(carboxy-ethylthio)propionyl, higher alkylsaturated and higher alkyl unsaturated dicarboxylic acids, from acomplex mixture comprising in addition to the compound of the generalformula 6-aminopenicillanic acid (6-APA) and optionally one or moreN-substituted β-lactam compounds, comprising the steps of: (a)acidifying the complex mixture to a pH below 6.5 and maintaining themixture below said pH at a temperature of between 10° C. and 150° C.;and/or (b) contacting the complex mixture with a carbon dioxide source;and (c) recovering the cephalosporanic acid compound of the formula (I)from the mixture obtained after steps (a) and/or (b).
 2. A methodaccording to claim 1, wherein in step (a) the temperature is keptbetween about 50° C. and about 130° C., preferably between 70 and 120°C., for between 10 seconds and about 1 day and the pH is kept at orbelow pH 4.5.
 3. A method according to claim 1 or 2, wherein thecompound has been produced by fermentation of a micro-organism capablethereof and wherein the complex mixture is a broth, a culture filtrateor any culture liquid derivable from the broth after fermentation.
 4. Amethod according to any one of claims 1 to 3, wherein the compound ofthe general formula is selected from the group consisting ofadipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA.
 5. A method according toany one of the previous claims, wherein step (c) is performed bysubjecting the mixture obtained after steps (a) and/or (b) tochromatography.
 6. A method according to claim 5, wherein chromatographyis adsorption chromatography, more preferably Hydrophobic InteractionChromatography.
 7. Use of chromatography in a process of recovering anN-substituted cephalosporin compound according to formula (I) inclaim
 1. 8. Use according to claim 7, wherein the chromatography isadsorption chromatography, preferably Hydrophobic InteractionChromatography.
 9. Use according to claim 8, wherein the chromatographyis performed using Simulating Moving Bed technology.
 10. A method forpreparing a compound of formula (II):

wherein R₀ is hydrogen or C₁₋₃ alkoxy; Y is CH₂, oxygen, sulphur, or anoxidised form of sulphur; R₁ is any of the groups selected from thegroup consisting of hydrogen, hydroxy, halogen, saturated orunsaturated, straight or branched alkyl (1-5 carbon atoms; optionallyreplaced by one or more heteroatoms), optionally substituted withhydroxy, halogen, aryl, alkoxy (1-3 carbon atoms), or acyl; alkoxy (1-3carbon atoms; optionally replaced by one or more heteroatoms),optionally substituted with hydroxy or halogen; or cycloalkyl (3-8carbon atoms) optionally substituted with hydroxy, halogen, amino; aryl;heteroaryl, comprising the steps of making a compound according toformula (I) using a process according to any one of claims 1 to 6;deacylating the compound of formula (I) to obtain a conversion solutionwhich comprises a compound according to formula (II).
 11. A methodaccording to claim 10, wherein the conversion solution further comprisesthe cleaved side chain designated R₂.
 12. The process of claim 10,wherein the deacylation is performed enzymatically using a dicarboxylacylase.
 13. A process according to any one of claims 10 to 12,comprising the further step of recovering the compound of formula (II)from the solution by crystallisation.
 14. A process according to claim13, wherein crystallisation is preceded by treatment of the solutionwith an agent selected from the group consisting of an adsorber resin,active coal, methanol, ethanol, (iso)propanol, isobutanol, n-butanol,acetone or a combination of any of the mentioned agents.
 15. A processaccording to claim 14, wherein at least an adsorber resin is usedselected from XAD16, XAD1600 and HP20.
 16. A method according to claim10 or 11, wherein the 6-aminopenicillanic acid (6-APA) level is 10 ppmor less with respect to the compound of formula (II).
 17. A processaccording to claim 11 or 12, wherein following the deacylation thesolution is treated to remove, at least partially, the cleaved sidechain represented by R₂.
 18. A process according to claim 17, whereinthe treatment to remove, at least partially, the cleaved side chain iscarried cut on the mother liquor obtained after crystallisation.
 19. Aprocess according to claim 18, wherein said treatment to remove, atleast partially, the cleaved side chain is followed by solubilisation ofthe crude crystals and recrystallisation of the compound of formula(II).
 20. A process according to claim 18, wherein crystallisation ispreceded by treatment of the solution with an agent selected from thegroup consisting of an adsorber resin, active coal, methanol, ethanol,(iso)propanol, isobutanol, n-butanol and acetone, or a combination ofany of these mentioned agents.
 21. A process according to any one ofclaims 17 to 20, wherein said treatment comprises subjecting theconversion solution, or the mother liquid, to membrane filtration at apH below 5, preferably below 4, more preferably near or below
 3. 22. Useof membrane filtration to remove a dicarboxylic acid from a mixturecomprising the dicarboxylic acid and a β-lactam antibiotic.
 23. Useaccording to claim 22, wherein the mixture is a mother liquid obtainedafter crystallisation of a compound of the formula (II).
 24. The useaccording to claim 22 or 23, wherein the filtration takes place at a pHof about 5 or less, preferably at pH 4 or less.
 25. The use according toclaim 22 to 24, wherein said filtration is by narofiltration at or belowpH 3.